mabs-4-419

© 2012 Landes Bioscience.

Do not distrib

ute.

www.landesbioscience.com mAbs 419

mAbs 4:4, 419-425; July/August 2012; © 2012 Landes Bioscience

Editor’s CornEr EditoriAL

At least 15 glyco-engineered antibodies are currently being evaluated in clinical studies. The next approval of a glyco-engineered antibody is likely to be obinu-tuzumab (GA101), the Roche-Glycart antibody that is currently in Phase 3 clini-cal trials. GA101 is a third-generation, humanized, glyco-engineered anti-CD20 IgG1 mAb that is undergoing evaluation for the potential treatment of B cell malig-nancies. GA101 induces 5- to 100-fold greater ADCC than observed upon treat-ment with rituximab. Another promising application of the Roche-Glycart technol-ogy is GA201 (RG7160), an epidermal growth factor receptor (EGFR)-targeting antibody, that could be indicated for the nearly 40% of colorectal patients with KRAS mutation who do not respond to cetuximab and panitumumab.1 GA201 is a dual-acting, humanized, IgG1 mAb that has been designed to provide enhanced ADCC activity and increased immune response in combination with signaling inhibition. Notably, there are a plethora of alternative production systems for glyco-optimized proteins, including yeast, duck, rat, algae, moss and tobacco. Last but not least, biobetter antibody versions of

Marketing approval of mogamulizumabA triumph for glyco-engineering

Alain Beck1,* and Janice M. reichert2

1Centre d’immunologie Pierre Fabre; saint-Julien-en-Genevois, France; 2Landes Bioscience; Austin, tX UsA

Keywords: ADCC, antibody, biobetters, biosimilars, CDC, follow-on biologicals, fucose, galactose, glyco-engineering, glycosylation, mannose, sialic acid

*Correspondence to: Alain Beck; Email: [email protected]: 05/15/12; Accepted: 05/15/12http://dx.doi.org/10.4161/mabs.20996

trastuzumab, cetuximab, rituximab and infliximab derived from these technolo-gies are also in development.

Current Production Systems for Approved IgGs

Chinese hamster ovary cells (CHO) and mouse myeloma cells (NS0, SP2/0) have become the gold-standard mammalian host cells for the production of therapeu-tic antibodies and Fc-fusion proteins that have already reached the market.2 Of the 28 mAbs marketed in the United States or European Union, 43% are produced in CHO cells, 50% in mouse-derived cells (18% in NS0, 25% in SP2/0 and 7% in hybridomas) and 7% in E. coli (non-gly-cosylated Fab).3 Most of these cell lines have been adapted to grow in suspension culture and are well-suited for reactor cul-ture, scale-up and large volume produc-tion (up to 20,000 L), with a productivity ranging from 1 to 8 g/L. Such manufac-turing scales are essential features for sup-plying antibodies used in chronic diseases for the world-wide market. Blockbuster antibodies are currently produced at a multi-ton scale per year. The main

glycoforms of antibodies and other gly-coproteins produced in these mammalian cell line systems are close to the human ones. But minor, non-human glycoforms also exist; these may be immunogenic, resulting in faster clearance if present in large amounts.

Antibody Glycosylation in Human Sera vs. Recombinant mAbs from

CHO, NS0 or SP2/0

The glycoforms identified on IgGs pro-duced from CHO cells are close to human ones except for the third GlcNac bisecting arm, which represents ~10% of human IgG glycoforms, and very low amounts of terminal N-acetylneuraminic acid (NANA; Fig. 1).4 Murine NS0 or SP2/0 cells produce mAbs exhibiting small amounts of glycoforms with addi-tional Gal α-1,3-gal and different sialic acids such as N-glycolylneuraminic acid (NGNA) instead of NANA. NGNA is the predominant sialic acid present in glycoproteins produced by mouse cells, but it appears only as traces in glycopro-teins expressed from CHO cells (Fig. 2).5 NGNA is reported to be immunogenic

therapeutic properties of antibodies frequently depend on the composition of their glycans. Most of the currently approved antibodies are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans, but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs. the recent approval in Japan of mogamulizumab (PotELiGEo®), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies. Mogamulizumab is a humanized mAb derived from Kyowa Hakko Kirin’s PotELLiGEnt® technology, which produces antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (AdCC) activity. the approval was granted March 30, 2012 by the Japanese Ministry of Health, Labour and Welfare for patients with relapsed or refractory CCr4-positive adult tcell leukemia-lymphoma.

© 2012 Landes Bioscience.

Do not distrib

ute.

420 mAbs Volume 4 issue 4

α-1,3-gal and NGNA were found only in the Fab moieties in contrast to the Fc frag-ment, for which only typical IgG G0F, G1F and G2F glycoforms were identified. In a recent report on cetuximab-induced anaphylaxis, pre-existing IgEs specific for this galactose-α-1,3-gal epitope were detected in patients treated with cetux-imab.7-9 Using a solid phase immunoas-say, these IgEs were found to bind to

glycoform (2–4%) on Asn297.5 A notable exception is cetuximab, which contains a second N-glycosylation site in its Fab portion on heavy chain Asn88. For the marketed version of cetuximab produced in SP2/0 cells, at least 21 different glyco-forms were identified with ~30% capped by at least one Gal α-1,3-gal residue, 12% capped by a NGNA residue and traces of oligomannose.6 Importantly, both Gal

in human, but, from a practical stand-point, the amount present in most of the NS0-produced mAbs is generally very low in the Fc part (~1–2%). No serious adverse events linked to these glycoforms were reported for the marketed NS0- and SP2/0-produced mAbs, e.g., palivizumab, which was first approved in 1998. The same stands for the mouse Gal α-1,3-gal residue, which is generally a very minor

Figure 1. igG antibody n-glycosylation.

Figure 2. Antibody glycosylation: human, recombinant and glyco-engineered.

© 2012 Landes Bioscience.

Do not distrib

ute.

www.landesbioscience.com mAbs 421

or follow-on biologicals with lower costs-of-goods than can be attained with mammalian cell lines. The glyco-engi-neering technology of the Pichia pasto-ris N-glycosylation pathway developed by GlycoFi allows production of human proteins with complex N-glycosylation modifications that are similar to the ones performed in human. Moreover, more homogeneous glycosylation patterns are observed, as opposed to the large hetero-geneity of glycan moieties that are found naturally in mammals or in other produc-tion systems such as CHO and NS0 cell lines. These properties, which are positive attributes when considering industrializa-tion of the manufacturing process, makes Pichia a very promising expression system to produce large-scale batches of therapeu-tics at a lower cost.

S. pombe and S. cerevisiae (Glycode technology). Glycode develops yeast strains that are deficient in high-man-nose-type glycosylation, and that express, upon stable integration, all enzymes needed to perform hybrid and complex-type N-glycosylation. Up to 30 different yeast strains that perform various steps of the mammalian glycosylation path-way are available. The feasibility of their technology was exemplified by the pro-duction of recombinant erythropoietin (EPO). An important advantage of this technology is based on the stability of the glyco-engineered strains. The selection of the desired knockout and knock-in yeast strains is based on auxotrophy selectable markers, which might be more stable than resistance markers classically used by oth-ers during scale-up and manufacturing process.

Filamentous fungi (Aspergillus niger and nidulans). Filamentous fungi (Aspergillus niger and nidulans) were also glyco-engineered by a approach similar to the one applied in Pichia pastoris by dele-tion of genes coding for fungal glycosyl-ation enzymes and introduction of genes necessary to produced humanized com-plex N-glycans.

Duck embryonic stem cells (EB66 cell line, Vivalis technology). Antibodies produced in EB66 cells display a natu-rally reduced fucose content that results in enhanced ADCC activity.23 A compara-tive N-linked oligosaccharide analysis of

has established a FUT8 knockout CHO cell line by gene targeting using a homolo-gous recombination technique.22 Except for the complete depletion of FUT8 expression, the properties of the estab-lished FUT8 knockout CHO cells were unaltered from those of the parent cells in terms of morphology, growth kinet-ics and productivity (POTELLIGENT® technology). Recombinant DNA-based glyco-engineering for increased antibody effector function was also achieved by overexpression of heterologous β1,4-N-acetylglucosaminyltransferase III (GnT-III) in antibody-producing cells,20 which is the Glycart-Roche technology. GnT-III catalyzes the addition of a bisecting GlcNAc to N-linked oligosaccharides. Once GnT-III adds a bisecting GlcNAc to an oligosaccharide, other central reac-tions of the biosynthetic pathway such as core-fucosylation and conversion of hybrid to complex glycans are blocked. Overexpression of GnTIII in antibody producing cells results in the formation of bisected, non-fucosylated oligosaccha-rides linked to the antibodies that mediate increased ADCC.

Cytotoxic enhancement for glyco-engi-neered mAbs with a bisecting GlcNAc or a depletion of fucose was not only dem-onstrated for CHO cells but also for a plethora of alternative systems like yeasts, baculovirus-infected insect cells, avian cells, YB2/0 rat cells, aquatic plants, moss and tobacco as illustrated in Table 1 and briefly discussed below.

Glyco-Engineered Antibodies with Humanized Glycoforms

in other Heterologous Expression Systems

Pichia pastoris (GlycoFi technology). GlycoFi’s glyco-engineering technol-ogy allows the generation of yeast strains capable of replicating the most essential steps of the N-glycosylation pathway found in mammals.13 Merck acquired GlycoFi in 2006 to synergize GlycoFi’s yeast glyco-engineering know-how and patent portfolio with Merck’s expertise in large-scale production of biologicals (e.g., Gardasil® human papillomavirus vaccine is produced in Saccharomyces cerevisiae) to produce enhanced biopharmaceuticals

SP2/0-produced cetuximab and F(ab)2 fragment, and not to the Fc fragment. Interestingly, no IgE immunoreactivity was found against a version of cetuximab produced in CHO (CHO-C225), which represents a simple way to produce a bio-better version of cetuximab.10,11

Effect of Glycosylation on Immunogenicity or Clearance

High mannose-type N-glycans contain from five to nine mannose residues and are found on antibodies produced in mam-malian cells,12 yeast,13 insect cells14 and plants,15 but only at a very low level in nor-mal human antibodies.16 High mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans.17,18 Several other glycoforms containing fucose or xylose moieties char-acteristic of mice, yeast or plant-derived glycoproteins are highly immunogenic in humans (Fig. 2). As a consequence, only mammalian-based production systems are used for the manufacturing of approved biopharmaceuticals, which need proper glycosylation. Nevertheless, tremendous efforts are made both in academic labs and in industry to engineer the glycosylation pathways of mammalian cells, yeasts, insect cells and plants to allow the pro-duction of recombinant proteins exhibit-ing human-like glycosylation.

Glyco-Engineered Antibodies in CHO Cells with Enhanced ADCC

ADCC is an important effector function, especially for human IgG1 mAbs devel-oped in oncology, when the major goal is to selectively destroy tumor cells.19 The pres-ence of a bisecting N-acetylglucosamine (GlcNAc) associated with the depletion in fucose residues (e.g., by genetic knockdown of α-1,6-fucosyltransferase) from oligo-saccharides in the conserved attachment region to Fcγ receptors results in an up to 100-fold increase in ADCC activity.20 The current CHO cell lines are not suitable for the production of completely defu-cosylated antibodies as they retain a high level of intrinsic α-1,6-fucosyltransferase (FUT8) enzyme activity, which is respon-sible for the core fucosylation of N-linked oligosaccharides.21 Kyowa Kirin Hakko

© 2012 Landes Bioscience.

Do not distrib

ute.

422 mAbs Volume 4 issue 4

other glycoforms and other product qual-ity attributes. The level of galactosylation could be controlled in one cell line from 3% to 23% by varying the concentration of these additives, and in a second cell line from 5% to 29%. This approach enabled design of a tailored process by adding the appropriate amount of chemicals to the culture medium to enhance the CDC type of effector functions.

Glyco-Engineered Antibodies with Enhanced Inflammatory

Properties

Sialylated glycans are known to be spe-cies-characteristic and essential compo-nents of glycoproteins as illustrated, for example, by the switch in specificity of avian influenza viruses hemagglutinins to human flu viruses (α-2–3 sialyl to α-2–6). Nevertheless, in contrast to other circu-lating glycoproteins (e.g., EPO), human IgGs are poorly sialylated. The same observation was reported for recombinant antibodies produced in eukaryotic cells. Interestingly, it was recently shown that antibody sialylation could suppress inflam-mation and reduce cytoxicity through the engagement of its Fc fragment with different Fc gamma receptors as demon-strated by Nimmerjahn and Ravetsch in several papers. For this purpose, in

fucosyltransferase) to block the process-ing of the corresponding non-mammalian sugar moieties.

Lemna minor. The Lemna minor expression system (LEX, an aquatic plant) enables rapid clonal expansion and secre-tion of mAbs at high yields (at least up to 300 g scale) with full containment and no risk of transmission of mam-malian pathogens. To avoid the expres-sion of immunogenic plant glycans, co-expression with single RNAi transcript to silence α-1,3-fucosyltransferase and β-1,2-xylosyltransferase was performed and shown to be stable at least for 3 y. As a proof-of-concept, increased ADCC activity and FcγRIIIa binding was dem-onstrated for an anti-CD30 mAb com-pared with the same antibody produced in CHO, as well as for an anti-CD20 mAb (BLX-300).25

Glyco-Engineered Antibodies with Enhanced CDC

Gramer et al. recently reported data show-ing that the combination of uridine, man-ganese chloride and galactose is useful for specifically affecting antibody galac-tosylation correlated with complement-dependent cytotoxicity (CDC) activity.26 This was demonstrated in a GS-CHO fed-batch process with minimal impact on

CHO- and EB66-produced rituximab was performed by mass spectrometry, as well as in vitro ADCC assays.

Plants. Plants are another attractive production system for recombinant pro-teins.24 A major concern is the presence of β-1,2-xylose (not present in human glycans) and α-1,3-fucose sugars (instead of α-1,6-fucose), which are allergenic epi-topes in human. The first generation of plant-derived antibodies (“plantibodies”) were investigated in early clinical trials a decade ago for topical applications (e.g., genital herpes, dental carries), but devel-opment of them was terminated. More recently, controlled glycosylation of anti-rabies antibodies was achieved in tobacco plants by expression of human light and heavy chains genetically fused to a Lys-Asp-Glu-Leu (“KDEL”) sequence at the C-terminal parts. Interestingly, this signal peptide allows the retention of the gly-coproteins in the endoplasmic reticulum and the biosynthesis of mainly oligoman-nose variants free of β-1,2-xylose and α-1,3-fucose.

Moss (Physcomitrella patens). Moss is alternatively proposed as a culture system for production of mAbs in photo-biore-actors. Non-immunogenic and ADCC-improved glycan patterns were obtained by targeted gene replacements of two moss enzymes (xylosyltransferase and

Table 1. selected antibody glyco-engineering technologies (biobetter or next-generation mAbs)

Company (country) Technology Cell line Antigen target Reference

GenmAb (nL) Galactosylation (CdC) ns0 (mice) Cd20 26

Glycode (Fr) GlycodExpress® Yeast www.glycode.fr

Glycotope (GE) Glycoexpress® Human EGFr, HEr2 28

Greenovation (GE) BryoMaster™Physcomitrella patensi

(Moss)29

Kyowa Hakko Kirin (JP) Lonza (UK) PotELLiGEnt® CHo (hamster) CCr4, Cd98, GM2, iL5 30

BioWa (JP), Lonza (UK) PotELLiGEnt®CHoK1sV CHo-Gs0

Cnrs (Fr)Baculovirus expressing Gnt-i, Gnt-ii

and β1–4 galactosyltransferaseBaculovirus/insect cell 14

LFB (Fr) EMABling® YB2/0 (rat) Cd20, rhesus d 31

Merck-GlycoFi (Us) Pichia pastoris (yeast) Cd20, HEr2 32

roche-Glycart (CH) GlycoMAb® CHo (hamster) Cd20, EGFr, HEr2, HEr3 33

synthon-Biolex (nL) LEX system Lemna minor (aquatic plant) Cd20, HEr2 25

siaMedExpress (Fr) siaMedExpress® CHo (hamster) not disclosed

Vivalis (Fr) EB66® EB66 (duck) Cd20 23

CCr4, C-C chemokine receptor type 4; Cd, cluster of differentiation; CHo, Chinese hamster ovary; EGFr, epidermal growth factor receptor; HEr, human epidermal growth factor receptor; iL, interleukin. Country abbreviations: CH, switzerland; GE, Germany; Fr, France; JP, Japan; nL, the netherlands; UK, United Kingdom; Us, United states

© 2012 Landes Bioscience.

Do not distrib

ute.

www.landesbioscience.com mAbs 423

B cell lymphoma, a Phase 2 study in adults with chronic lymphocytic leukemia, and a Phase 1 study in adults with advanced B cell malignancies. A Phase 1/2 study [NCT01585766] of MEDI-551 in adults with relapsing-remitting multiple scle-rosis was planned but not yet recruiting patients as of mid-May 2012. BIW-8962 was undergoing evaluation as monother-apy in a Phase1/2 study [NCT00775502] of patients with previously treated mul-tiple myeloma, but the study was termi-nated due to lack of efficacy. The three mAbs at Phase 1 are undergoing evalua-tion as therapy for patients with advanced solid tumors.

Three GlycoMabTM-derived mAbs (obinutuzumab, GA201, RG7116) are in clinical study. Obinutuzumab, which targets CD20, is undergoing evalua-tion in four Phase 3 studies, four Phase 2 studies, and two Phase 1 studies, all of which include patients with hematologi-cal malignancies. The anti-EGFR GA201 is currently being evaluated in a Phase 2 study of patients with non-small cell lung cancer, a Phase 2 study of patients with colorectal cancer and a Phase 1 study of patients with head and neck squamous cell

marketing and six POTELLIGENT®-derived mAbs are in clinical studies. Mogamulizumab (POTELIGEO®) was approved in Japan in March 2012 as a treatment for patients with relapsed or refractory CCR4-positive T cell leukemia-lymphoma. Kyowa Hakko Kirin is also evaluating mogamulizumab in patients with peripheral T-cell lymphoma (PTCL) or cutaneous T-cell lymphoma. The mAb is licensed to Amgen for development in non-cancer indications. In December 2011, Amgen initiated a Phase 1 study of mogamulizumab in adults with asthma.

Of the six POTELLIGENT®-derived mAbs in clinical studies, three (ben-ralizumab, MEDI-551, BIW-8962) have advanced to Phase 2 studies and three (KHK2898, KHK2804, KHK2866) are in Phase 1 studies. Benralizumab is an IgG1k mAb that targets interleukin (IL)-5 receptor α chain; it is undergoing evaluation as a treatment for asthma and for moderate-to-severe chronic obstruc-tive pulmonary disease and sputum eosinophilia. MEDI-551, which targets CD19 on B cells, is currently in a Phase 1/2 study of patients with scleroderma, a Phase 2 study in adults with diffuse large

vitro desialylation was achieved by anti-body incubation with neuraminidase and the anti-inflammatory properties of the IgGs were lost. On the other hand, over-sialylated antibodies were obtained by affinity-chromatography purification with agarose-bound lectins and shown to have enhanced anti-inflammatory activi-ties. Alternatively, terminally sialylated recombinant antibodies could be obtained in engineered yeast and for this purpose the GlycoFi technology looks very promis-ing.27 Higher-level antibody sialylation is associated with reduced ADCC, which is another indication of the pharmacological importance of these residues, as well as of the fine structural tuning of glycosylation, that can be achieved by the GlycoFi or SiaMedExpress technologies.

Glyco-Engineered Antibodies in Clinical Trials

To our knowledge, a total of 16 mAbs derived from four different glyco-engi-neering approaches have entered clinical studies (Table 2). One mAb derived from Kyowa Hakko Kirin’s POTELLIGENT® technology has been approved for

Table 2. Glyco-engineered antibodies in clinical study

Company Name (INN or company code) Target Format; glyco-engineering technology Status

Kyowa Hakko Kirin Mogamulizumab, KW0761, AMG761 CCr4 Humanized igG1; PotELLiGEnt® Approved in Japan

Kyowa Hakko Kirin/Medimmune

Benralizumab, KHK4563, MEdi-563 iL5 receptor Humanized igG1; PotELLiGEnt® Phase 2

Medimmune/Kyowa Hakko Kirin

MEdi-551 Cd19 Humanized igG1; PotELLiGEnt® Phase 2

Kyowa Hakko Kirin BiW-8962 GM2 ganglioside Humanized igG1/3; PotELLiGEnt® Phase 2

Kyowa Hakko Kirin KHK2898 Cd98 Human; PotELLiGEnt® Phase 1

Kyowa Hakko Kirin KHK2804, CEP-37250 tumor glycan Humanized; PotELLiGEnt® Phase 1

Kyowa Hakko Kirin KHK2866 HB-EGF Human; PotELLiGEnt® Phase 1

Genentech/roche obinutuzumab, GA101, ro5072759 Cd20 Humanized igG1; GlycoMabtM Phase 3

Genentech/roche GA201, rG7160, ro5083945 EGFr Humanized igG1; GlycoMabtM Phase 2

Genentech/roche rG7116, ro5479599 HEr3 Humanized; GlycoMabtM Phase 1

Glycotope Gt-MAB2.5GEX, MUC-1 Humanized; GlycoExpresstM Phase 1

Glycotope Gt-MAB5.2GEX, CetuGEX EGFr Chimeric; GlycoExpresstM Phase 1

Glycotope Gt-MAB7.3GEX, trasGEX HEr2 Human; GlycoExpresstM Phase 1

Life science Pharmaceuticals

Ecromeximab, KW-2871 Gd3 Chimeric igG1; YB2/0 cell line Phase 2

LFB roledumab, LFB-r593 rhesus d Human igG1; YB2/0 cell line Phase 2

LFB/tG therapeutics Ublituximab, LFB-r603, tGtX-1101 Cd20 Chimeric igG1; YB2/0 cell line Phase 1

Based on data available as of May 15, 2012. Abbreviations: CCr4, C-C chemokine receptor type 4; Cd, cluster of differentiation; CHo, Chinese hamster ovary; EGFr, epidermal growth factor receptor; HB-EGF, heparin-binding EGF-like growth factor; HEr, human epidermal growth factor receptor; iL, interleukin; MUC, mucin

© 2012 Landes Bioscience.

Do not distrib

ute.

424 mAbs Volume 4 issue 4

15. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J. GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 2012; 30:434-9; PMID:21856403; http://dx.doi.org/10.1016/j.bio-techadv.2011.08.007.

16. Goetze AM, Schenauer MR, Flynn GC. Assessing monoclonal antibody product quality attribute criti-cality through clinical studies. MAbs 2010; 2:500-7; PMID:20671426; http://dx.doi.org/10.4161/mabs.2.5.12897.

17. Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 2011; 21:949-59; PMID:21421994; http://dx.doi.org/10.1093/glycob/cwr027.

18. Alessandri L, Ouellette D, Acquah A, Rieser M, LeBlond D, Saltarelli M, et al. Increased serum clear-ance of oligomannose species present on a human IgG1 molecule. MAbs 2012; In press.

19. Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeu-tic antibodies. Nat Rev Immunol 2010; 10:345-52; PMID:20414207; http://dx.doi.org/10.1038/nri2747.

20. Umaña P, Jean-Mairet J, Moudry R, Amstutz H, Bailey JE. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 1999; 17:176-80; PMID:10052355; http://dx.doi.org/10.1038/6179.

21. Kubota T, Niwa R, Satoh M, Akinaga S, Shitara K, Hanai N. Engineered therapeutic antibodies with improved effector functions. Cancer Sci 2009; 100:1566-72; PMID:19538497; http://dx.doi.org/10.1111/j.1349-7006.2009.01222.x.

22. Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Iida S, Nakano R, et al. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosyl-ated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 2004; 87:614-22; PMID:15352059; http://dx.doi.org/10.1002/bit.20151.

23. Olivier S, Jacoby M, Brillon C, Bouletreau S, Mollet T, Nerriere O, et al. EB66 cell line, a duck embry-onic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity. MAbs 2010; 2:405-15; PMID:20562528.

24. Saint-Jore-Dupas C, Faye L, Gomord V. From planta to pharma with glycosylation in the toolbox. Trends Biotechnol 2007; 25:317-23; PMID:17493697; http://dx.doi.org/10.1016/j.tibtech.2007.04.008.

25. Gasdaska JR, Sherwood S, Regan JT, Dickey LF. An afucosylated anti-CD20 monoclonal antibody with greater antibody-dependent cellular cytotox-icity and B-cell depletion and lower complement-dependent cytotoxicity than rituximab. Mol Immunol 2012; 50:134-41; PMID:22305040; http://dx.doi.org/10.1016/j.molimm.2012.01.001.

26. Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, et al. Modulation of antibody galac-tosylation through feeding of uridine, manganese chlo-ride and galactose. Biotechnol Bioeng 2011; 108:1591-602; PMID:21328321; http://dx.doi.org/10.1002/bit.23075.

27. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 2006; 313:1441-3; PMID:16960007; http://dx.doi.org/10.1126/science.1130256.

28. Lugovskoy AA, Reichert JM, Beck A. 7th Annual European Antibody Congress 2011: November 29–December 1, 2011, Geneva, Switzerland. MAbs 2012; 4:134-52; PMID:22453093; http://dx.doi.org/10.4161/mabs.4.2.19426.

29. Decker EL, Reski R. Glycoprotein production in moss bioreactors. Plant Cell Rep 2012; 31:453-60; PMID:21960098; http://dx.doi.org/10.1007/s00299-011-1152-5.

many more in preclinical development, the future of glyco-engineering looks bright indeed.

References1. Paz-Ares LG, Gomez-Roca C, Delord JP, Cervantes

A, Markman B, Corral J, et al. Phase I pharmacoki-netic and pharmacodynamic dose-escalation study of RG7160 (GA201), the first glycoengineered mono-clonal antibody against the epidermal growth factor receptor, in patients with advanced solid tumors. J Clin Oncol 2011; 29:3783-90; PMID:21900113; http://dx.doi.org/10.1200/JCO.2011.34.8888.

2. Beck A, Wagner-Rousset E, Bussat MC, Lokteff M, Klinguer-Hamour C, Haeuw JF, et al. Trends in glyco-sylation, glycoanalysis and glycoengineering of thera-peutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 2008; 9:482-501; PMID:19075687; http://dx.doi.org/10.2174/138920108786786411.

3. Reichert JM. Marketed therapeutic antibodies compen-dium. MAbs 2012; 4:413-5; PMID:22531442; http://dx.doi.org/10.4161/mabs.19931.

4. Jefferis R. Isotype and glycoform selection for anti-body therapeutics. Arch Biochem Biophys 2012; In press; PMID:22465822; http://dx.doi.org/10.1016/j.abb.2012.03.021.

5. Raju TS, Jordan RE. Galactosylation variations in marketed therapeutic antibodies. MAbs 2012; 4:385-91; PMID:22531450; http://dx.doi.org/10.4161/mabs.19868.

6. Sundaram S, Matathia A, Qian J, Zhang J, Hsieh MC, Liu T, et al. An innovative approach for the charac-terization of the isoforms of a monoclonal antibody product. MAbs 2011; 3:505-12; PMID:22123057; http://dx.doi.org/10.4161/mabs.3.6.18090.

7. Mariotte D, Dupont B, Gervais R, Galais MP, Laroche D, Tranchant A, et al. Anti-cetuximabIgE ELISA for identification of patients at a high risk of cetuximab-induced anaphylaxis. MAbs 2011; 3:396-401; PMID:21654207; http://dx.doi.org/10.4161/mabs.3.4.16293.

8. Daguet A, Watier H. 2nd Charles Richet et Jules Héricourt workshop: therapeutic antibodies and ana-phylaxis; May 31–June 1, 2011, Tours, France. MAbs 2011; 3:417-21; PMID:21857165; http://dx.doi.org/10.4161/mabs.3.5.17485.

9. Lammerts van Bueren JJ, Rispens T, Verploegen S, van der Palen-Merkus T, Stapel S, Workman LJ, et al. Anti-galactose-α-1,3-galactose IgE from allergic patients does not bind α-galactosylated glycans on intact therapeutic antibody Fc domains. Nat Biotechnol 2011; 29:574-6; PMID:21747378; http://dx.doi.org/10.1038/nbt.1912.

10. Wang C, He X, Zhou B, Li J, Li B, Qian W, et al. Phase 1 study of anti-epidermal growth factor receptor monoclonal antibody in patients with solid tumors. MAbs 2011; 3:67-75; PMID:21051930; http://dx.doi.org/10.4161/mabs.3.1.14021.

11. Beck A, Cianférani S, Van Dorsselaer A. Biosimilar, biobetter and next generation antibody characterization by mass spectrometry. Anal Chem 2012; 84:4637-46; PMID:22510259; http://dx.doi.org/10.1021/ac3002885.

12. Pacis E, Yu M, Autsen J, Bayer R, Li F. Effects of cell culture conditions on antibody N-linked glycosylation-what affects high mannose 5 glycoform. Biotechnol Bioeng 2011; In press; PMID:21557201; http://dx.doi.org/10.1002/bit.23200.

13. Beck A, Cochet O, Wurch T. GlycoFi’s technology to control the glycosylation of recombinant therapeutic proteins. Expert Opin Drug Discov 2010; 5:95-111; http://dx.doi.org/10.1517/17460440903413504.

14. Cérutti M, Golay J. Lepidopteran cells, an alternative for the production of recombinant antibodies? MAbs 2012; 4:294-309; PMID:22531440; http://dx.doi.org/10.4161/mabs.19942.

carcinoma. RG7116, which targets human epidermal growth factor receptor (HER)-3, is in a Phase 1 dose-escalation study in patients with HER3-positive solid tumors.

Three GlycoExpressTM-derived mAbs (GT-MAB2.5GEX, GT-MAB5.2GEX, GT-MAB7.3GEX) are in Phase 1 stud-ies. The safety and tolerability of GT-MAB2.5GEX, which targets MUC1, is being evaluated in a dose escalation study in patients with advanced MUC1-positive solid malignancies. Anti-EGFR GT-MAB5.2GEX and anti-HER2 GT-MAB7.3GEX are undergoing evalu-ation in Phase 1 studies of patients with EGFR-positive and HER2-positive solid tumors, respectively. The estimated study completion date for all three of these Phase 1 studies is June 2012.

Three mAbs produced in YB2/0 cells, and therefore with low fucose content, are currently in Phase 2 clinical studies. The safety and effectiveness of ecromeximab, developed by Kyowa Hakko and licensed by Life Science Pharmaceuticals, is being evaluated in a Phase 2 study of patients with metastatic melanoma. LFB is develop-ing two low-fucose mAbs, roledumab and ublituximab (EMABLING technology). Anti-rhesus (Rh) D roledumab was evalu-ated in a Phase 2 study [NCT00952575] designed to demonstrate the ability of LFB-R593 to effectively eliminate exog-enously-administered RhD-positive red blood cells from the circulation of an RhD-negative individual, thereby preventing RhD-alloimmunization. Ublituximab, which targets CD20, was evaluated in a Phase 1 study [NCT01098188] of patients with chronic lymphocytic leukemia. TG Therapeutics, Inc., licensed the world-wide commercial rights to ublituximab in March 2012.

Future of Glyco-Engineering

Research done during the 1990s and 2000s on the glyco-engineering of anti-bodies has yielded a wide variety of approaches to production and a growing pipeline of these molecules. Thus, the technology is now delivering on the prom-ise of therapeutic mAbs with improved properties compared with first-generation versions. With one glyco-engineered mAb approved, at least 15 in the clinic and

© 2012 Landes Bioscience.

Do not distrib

ute.

www.landesbioscience.com mAbs 425

33. Sehn LH, Assouline SE, Stewart DA, Mangel J, Gascoyne RD, Fine G, et al. A phase 1 study of obinu-tuzumab induction followed by 2 years of maintenance in patients with relapsed CD20-positive B-cell malig-nancies. Blood 2012; 119:5118-25; PMID:22438256; http://dx.doi.org/10.1182/blood-2012-02-408773.

32. Zhang N, Liu L, Dumitru CD, Cummings NR, Cukan M, Jiang Y, et al. Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclini-cal study. MAbs 2011; 3:289-98; PMID:21487242; http://dx.doi.org/10.4161/mabs.3.3.15532.

30. Yamane-Ohnuki N, Satoh M. Production of thera-peutic antibodies with controlled fucosylation. MAbs 2009; 1:230-6; PMID:20065644; http://dx.doi.org/10.4161/mabs.1.3.8328.

31. Oflazoglu E, Audoly LP. Evolution of anti-CD20 monoclonal antibody therapeutics in oncology. MAbs 2010; 2:14-9; PMID:20081379; http://dx.doi.org/10.4161/mabs.2.1.10789.